2&#39; -0-Trisubstituted silyloxymethyl-ribonucleoside-derivative and method for preparing the same

ABSTRACT

The invention provides ribonucleoside derivatives with novel protecting groups and methods for the preparation of such ribonucleoside derivatives. The general formula (I)  
                 
 
of the ribonucleoside derivatives is: wherein R 1  is a base of the purine- or pyrimidine-family or a derivative of such a base or any other residue with serves as a nucleobase surrogate, R 2  is a proton or a substituted derivative of phosphonic acid, R 3  is a proton or a protection-group for the oxygen atom in 5′-position, R 4 , R 5  and R 6  are independently alkyl or aryl or a combination of alkyl and aryl or heteroatom, R 4 , R 5  or R 6  may also be cyclically connected to each other; and wherein at least one of the R 4 , R 5  or R 6  substituents comprises a tertiary C-atom or a heteroatom vicinal to the Si-atom.

FIELD OF THE INVENTION

The invention is in the field of nucleic acid chemistry and concernsmethods for the preparation and use of ribonucleoside derivatives withnovel protecting groups. The inventive compounds are particularlyadapted for the automated preparation of oligoribonucleotides.

BACKGROUND OF THE INVENTION

Applications for synthetic nucleic acids are numerous and key for theunderstanding of biological processes. Among these applications, the useof synthetic oligonucleotides for the specific down-regulation ofproteins by specific hybridisation in the cell of the syntheticoligonucleotide to a mRNA is known as an antisense mechanism and hasbeen widely described (1). More recently, RNA interference, a techniqueusing dsRNA known as siRNA, has been successfully used to inhibit thetranslation of mammalian mRNA to its protein (2). The great promise ofRNA technology has created a need for the development of efficient andcost effective preparation of synthetic oligoribonucleotides.

Oligoribonucleotide synthesis is more challenging thanoligodeoxynucleotide synthesis, mainly because of the 2′-OH group whichis present in ribonucleic acids, but not in deoxyribonucleic acids.Chemical synthesis of oligoribonucleotides is normally based on aprotected ribonucleoside derivative immobilized on a solid phase towhich further protected ribonucleotide derivatives are coupled inconsecutive steps of one synthesis cycle each until the desired lengthof chain is achieved. To ensure an efficient synthesis and to avoid RNAdegradation during the preparation process, the protecting groupstrategy for the 2′-OH group should be perfectly orthogonal with that ofother protecting groups and the protecting group should be removed aslate as possible in the process. So far, mainly the following types ofprotection groups have been used to protect the 2′-OH group:

2′-O-TBDMS chemistry is the commonly used protecting group for RNAsynthesis (3). It is orthogonal with other protecting groups. However,2′-3′ phosphoryl migration during oligoribonucleotide synthesis has beenreported (4). Moreover, steric hindrance of t-butyldimethylsilyl groupclose to reactive phosphoramidite significantly diminishes couplingefficiency. The latter limitation can be reduced but at the price oflonger coupling times, the use of higher molar excesses of reagents andspecial phosphoramidite activators like 5-(Benzylmercapto)-1H-tetrazolefor instance (5).

2′-O-ACE chemistry has been described by Caruthers et al. (6) as analternative to TBDMS chemistry. There, 2′-OH is protected by an acidlabile orthoester. As compared to TBDMS, lower hindrance of thatprotecting group allows higher coupling rates. The acid lability of 2′orthoester requires a non-acid labile temporary protection of the 5′-OH.This was accomplished with trisubstituted silyl groups which are removedat the end of each coupling cycle by a fluoride-containing solution.Consequently, reagents for the preparation of 2′-O-ACE building blockshave to be scaled up specifically. Such 5′ deprotection may in somecases be problematic: it requires a dedicated synthesizer resistant tofluoride ions and the use of the commonly used silica based supports(like Controlled Pore Glass) is not possible. 2′-O-TOM chemistry hasbeen reported by Pitsch et al. (6). As for 2′-OTBDMS protecting groupstrategy, 2′-O-TOM protecting group is removed upon treatment withfluoride ions. Originally, it was developed to allow the synthesis oflong RNA without the 2′-3′ phosphoryl migration observed with2′-O-TBDMS. In this case, due to the acetal nature of the bond betweennucleoside and protection-group, no migration of the protection-group toa different position in the ribonucleoside-derivative, in particular tothe neighbouring 3′-O-position, can occur. Such isomerization is a wellknown problem in the synthesis of the conventional2′-O-silyl-substituted RNA-units.

An additional important advantage of this protecting group is the lowerhindrance of the protecting group due to the acetal spacer between2′-oxygen and the bulky triisopropylsilyl group.

Both 2′-O-TOM and 2′-O-ACE are affording coupling yields approachingthose observed in oligodeoxynucleotide synthesis. Satisfying couplingyields are also obtainable with 2′-O-TBDMS chemistry but at price of useof unusual activators or of higher molar excesses of building blocks. Inall cases, the building blocks mentioned are contributing to a largeextent to the manufacturing costs of oligoribonucleotides. Secondly,post-synthetic processing of oligoribonucleotides is more demanding ascompared to processing of oligodeoxyribonucleotides. The latter aspectcan be of special importance when high-throughput demand has to besatisfied.

There is a need for improved RNA building blocks which are easilyaffordable and allowing an easier post-synthetic processing.

Substituted silyloxymethyl groups have been used as protecting groups ofhydroxyl groups in the past (7,8). Steric hindrance of substituents onSi atom modulates stability and removal conditions of the protectinggroup. For instance, silyloxymethyl bearing tertiary carbons vicinal tothe Si atom have been reported (7), in these cases, yields for removalof protecting groups were suboptimal as compared to those usuallyobserved with TBDMS protection, and apparently not suited for solidphase oligoribonucleotide synthesis.

The present invention now provides novel and improved groups for theprotection at the 2′-OH position of ribonucleosides derivatives that areparticularly suited for automated oligoribonucleotide solid phasesynthesis. A new procedure for the preparation of these building blocksis provided. Finally, the use of these building blocks in RNA solidphase synthesis is disclosed.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides ribonucleoside-derivativesof the formula

wherein

-   -   R₁ is a base of the purine- or pyrimidine-family or a derivative        of such a base or any other residue which serves as a nucleobase        surrogate,    -   R₂ is a proton or a substituted derivative of phosphonic acid,    -   R₃ is a proton or a protection-group for the oxygen atom in        5′-position,    -   R₄, R₅ and R₆ are independently alkyl or aryl or a combination        of alkyl and aryl or heteroatom, R₄, R₅ or R₆ may also be        cyclically connected to each other; and    -   wherein at least one of the R₄, R₅ or R₆ substituents comprises        a tertiary C-atom or a heteroatom vicinal to the Si-atom.

In a preferred aspect, the substituent comprising the tertiary C-atomvicinal to the Si-atom comprises from 4 to 24 C-atoms, more preferablyfrom 5 to 24 C-atoms and yet more preferably from 6 to 24 C-atoms. In amore preferred aspect, the substituent comprising the tertiary C-atomvicinal to the Si-atom Is an alkyl-substituent selected from the groupconsisting of tert-butyl, tert-pentyl, tert-hexyl, tert-heptyl,tert-octyl, tert-nonyl, tert-decyl, tert-undecyl, tert-dodecyl. In aanother preferred aspect, the substituent comprising the tertiary C-atomvicinal to the Si-atom is selected from the group of 1,1-dimethyl ethyl,1,1-dimethyl-propyl, 1,1-dimethyl-butyl, 1,1-dimethyl-pentyl,1,1-dimethyl-hexyl, thexyl(1,1,2-trimethyl-propyl),1,1,2-trimethyl-butyl, 1,1,2-trimethyl-pentyl, 1,1,2-trimethyl-hexyl,1,1,2,2 tetramethyl-propyl, 1,1,2,2-tetramethyl-butyl. In a morepreferred embodiment, the substituents of the above groups comprises atleast 5 C-atoms, more preferably at least 6 C-atoms.

In a related aspect, the present invention providesribonucleoside-derivatives wherein the substituent vicinal to theSi-atom comprises a substituted heteroatom. In a preferred aspect, thesubstituent vicinal to the Si-atom comprises a substituted bivalentheteroatom, in a more preferred aspect this substituent is oxygen.

In another aspect, the present invention provides a method for thepreparation of a ribonucleoside-derivatives, comprising reacting anucleoside with the formula

where R₁ and R₃ are as defined as above, with a silyloxymethylderivative of the formula

wherein Y is a suitable leaving group and wherein R₄, R₅ and R₆ areindependently alkyl or aryl or a combination of alkyl and aryl or aheteroatom, R₄, R₅ or R₆ may also be cyclically connected to each other.In a preferred embodiment Y is halogen. In another preferred embodiment,R₄, R₅ and R₆ together comprise between 6 and 30 carbon atoms. In afurther preferred embodiment, R₄, R₅ and R₆ comprise at least onesubstituted heteroatom vicinal to Si atom, which is preferably abivalent atom, more preferably oxygen. The ribonucleoside derivative mayfurther be substituted on the oxygen in 3′-position with a groupcomprising of a derivative of phosphonic acid.

Another aspect of the present invention provides a method for thepreparation of a ribonucleoside-derivative, comprising reacting aribonucleoside derivative with the formula

upon an electrophilic activation with a compound of formula:

wherein R₁ is defined as above and R₇ is a alkyl- or aryl-group, oralkyl-aryl-group,

wherein R₂ is a protecting group,

wherein R₃ is a protecting group,

wherein R₄, R₅ and R₆ are defined as above.

In a preferred embodiment, the ribonucleoside derivative is furthersubstituted on the oxygen in 3′-position with a group comprising of aderivative of phosphonic acid.

DETAILED DESCRIPTION OF THE INVENTION ABBREVIATIONS

-   TBDMS t-butyldimethylsilyl-   ACE -bis[2-(acetyloxy)ethoxy]methyl-   TOM (triisopropylsilyl)oxymethyl-   THEX [((1,1,2-trimethyl-propyl)-dimethylsilyl)]-oxymethyl-   DCA Dichloroacetic acid-   dsRNA Double-stranded RNA-   siRNA Small interfering RNA

The present invention relates to 2′-O-silyloxymethylribonucleotide-derivatives for application in the chemical synthesis ofribonucleic acids comprising a D or L-ribose unit having the followinggeneral structural formula:

whereby

R₁ is a base of the purine- or pyrimidine-family or a derivative of sucha base or any other residue which serves as a nucleobase surrogate, R₂is a proton or a substituted derivative of phosphonic acid, R₃ is aproton or a protection-group for the oxygen atom in 5′-position, and R₄,R₅ and R₆ are independently alkyl- or aryl-groups or a alkyl-aryl-group.R₄, R₅ or R₆ may also be cyclically connected to each other.

The protection group R₃ in 5′-O-position is e.g. a monomethoxytrityl- ordimethoxytrityl-group or a different, suitable group which is removedfrom the growing sequence during chain building such freeing a bondingposition for coupling the next unit to be added to the chain.

The base component R₁ of the ribonucleoside derivative is preferably abase of the purine or pyrimidine family, e.g. one of the five nucleobaseadenine, cytosine, thymine, uracil, guanine or a derivative thereof, orany other residue which serves as a nucleobase surrogate. It can beprotected by an acyl-substituent which can be removed after chaincreation.

In the 3′-O-position, R₂ is a derivative of phosphonic acid, such as anN,N- and O-substituted phosphoramidite group, whereby the N-substituentsare alkyl- or aryl-groups which can be further substituted and /orcyclically connected to each other. By activation of the nitrogen of thedisubstitued amino-group the phosphorus centre is activated for couplingthe unit to a growing chain.

This invention now provides new and advantageous 2′-O-silyloxymethylprotecting groups wherein R₄, R₅ or R₆ is independently an alkyl- oraryl-substituent, or an alkyl-aryl- or aryl-alkyl or a substitutedheteroatom substituent, and

wherein at least one of the R₄, R₅ or R₆ substituents comprises aheteroatom or a tertiary C-atom as can be represented by the formula

wherein R′, R″ and R′″ are alkyl- or aryl, or an alkyl-aryl-substituent-or aryl-alkyl or a substituted heteroatom, and wherein R′, R″ and R′″are not H. R′, R″ and R′″ may be the same or different, preferred aresubstituents comprising 1 to 12 C-atoms, preferably 1 to 6 C-atoms andmore preferably are 1 to 4 C-atoms. R′, R″ and R′″ may also becyclically connected to each other, for instance R′ may be cyclicallyconnected to R″ or R′″, or R″ may be cyclically connected to R′″. In apreferred embodiment, two of the substituents are identical and comprisefrom 1 to 6 C-atoms, preferably from 1 to 4 C-atoms. The thirdsubstituent comprises preferably at least 3 C-atoms, preferred are from3 to 12 C-atoms, more preferred are from 3 to 6 C-atoms.

Thus, in one embodiment at least one of the substituents R₄, R₅ and/orR₆ is (C₄to C₂₄)-tertiary-alkyl and/or aryl, preferably (C₅ toC₁₈)-tertiary-alkyl and/or aryl, more preferably (C₆ toC₁₂)-tertiary-alkyl and/or aryl, wherein the tertiary C-atom is vicinalto the Si-atom. Without intending to be limited to these groups,examples of such substituents may comprise for instance tert-butyl,tert-pentyl, tert-hexyl, tert-heptyl, tert-octyl, tert-nonyl,tert-decyl, tert-undecyl, tert-dodecyl, thexyl(1,1,2-trimethyl-propyl),1,1,2-trimethyl-butyl, 1,1,2-trimethyl-pentyl, 1,1,2-trimethyl-hexyl,1,1,2,2tetramethyl-propyl, 1,1,2,2-tetramethyl-butyl. In a preferredembodiment, the substituent is tert-pentyl or higher, in a morepreferred embodiment the substituent is tert-hexyl or higher. Morepreferred examples comprise e.g. 1,1dimethyl-ethyl, 1,1-dimethyl-propyl,1,1-dimethyl-butyl, 1,1-dimethyl-pentyl, 1,1-dimethyl-hexyl,1,1,2-trimethyl-propyl, 1,1,2-trimethyl-butyl, 1,1,2-trimethyl-pentyl,1,1,2-trimethyl-hexyl, 1,1,2,2 tetramethyl-propyl,1,1,2,2-tetramethyl-butyl. In another embodiment R₄, R₅ and/or R₆comprise a heteroatom.

The substituent(s) which do not comprise a tertiary C-atom may beidentical or different substituents. These substituents are preferablyalkyl- or aryl-substituents, or alkyl-aryl-substituents. Preferred aresubstituent comprising from 1 to 12 C-atoms, preferably from 1 to 8C-atoms, more preferably from 1 to 4 C-atoms. Without intending to belimited to these groups, examples of such substituents may comprise forinstance methyl, ethyl, propyl, butyl, pentyl, hexyl, i-propyl,sec-butyl, isobutyl, sec-pentyl.

In another embodiment, R₄, R₅ and/or R₆ comprise a substitutedheteroatom like for instance silicon, germanium, tin, lead, nitrogen,oxygen, sulfur such as for instance can be represented for a“four-valent” heteroatom by the formula:

wherein X is Si or Ge, Sn or Pb and wherein R′, R″ and R′″ are definedas for formula 2, and R₄ and R₆ are defined as above. Any of thesubstituents R₄, R₅ or R₆ may comprise the heteroatom vicinal to theSi-atom, preferred only one as is exemplified in formula 3. For“bivalent” heteroatoms like oxygen, or for trivalent heteroatoms likenitrogen formula 3 may be adapted accordingly as a person of skill inthe art would readily recognize. In a preferred embodiment of thepresent invention, X is oxygen.

The compound may be prepared by methods known in the art such as via theorganometallic route. This route is described for instance in WO99/09044(6). The reaction 4→5a/5b→6 shows an example of the preparation of acompound of the present invention via the organometallic route. Briefly,a ribonucleoside protected at the 5′-O is reacted with e.g.chloromethyl[dimethyl-(1,1,2-trimethylpropyl)silyl]ether (or THEX—Cl) inthe presence of a suitable organometallic salt, such as for instancedibutyltindichloride or dibutyltinoxide. THEX—Cl itself is prepared in amanner similar to TOM-Cl according to a published procedure (7).However, unlike TOM-Cl, the preparation of THEX—Cl does not require afinal distillation step of the reagent prior to the reaction with theribonucleoside which represents a significant simplification. In thereaction of THEX—Cl with a 5′-O-protected ribonucleoside a mixture of2′- and 3′-protected ribonucleosides is obtained, wherefrom the2′-substituted ribonucleoside is purified by for instancechromatographic means. In a subsequent step the 3′-OH group of thepurified compound 5a is converted to the phosphoramidite 6 according tomethods known in the art (Sinha, N. D. et al., Tetrahedron Lett. 1983,24, 5843; Sinha, N. D. et al., Nucleic Acid Res. 1984, 12, 4539).

This method requires the use of substituted silyloxymethyl etherswherein substituents contain alkyl, aryl, or arylalkyl groups and whichcan be cyclically connected. In a further embodiment, this method isapplicable to silyloxymethylethers wherein at least one of thesubstituents contains at least one substituted heteroatom like forinstance silicon, germanium, tin, lead, nitrogen, oxygen, sulfur

The reaction may be carried out in solution or on solid phase or byusing polymer supported reagents. The solvent can be a hydrocarbonsolvent, ethereal solvent, nitrile solvent, chlorinated solvent,heterocyclic solvent, sulfoxide solvents, etc. Specific examples ofsuitable solvents include pyridine, N,N-dimethylformamide (DMF),tetrahydrofuran (THF), dimethylsulfoxide (DMSO), acetonitrile,dichloroethane and methylene chloride. Preferrably, dichloroethane isused.

Although the reaction may be carried out at room temperature, it mayalso be carried out at a temperature range of 0 to 150° C. preferably at10 to 100° C.

In a further aspect of the present invention, the compounds of theinvention are prepared by a new and superior method as exemplified bythe reaction 7→8→9→10→6.

This method allows the selective introduction of 2′-OH protecting groupsvia first introducing a 2′-O-alkylthiomethyl, arylthiomethyl,alkylarylthiomethyl or arylalkylthiomethyl group. It avoids theunselective step described in the organometallic route. This new methodis generally applicable for the introduction of oxymethyl derivativesselectively on 2′-OH group of ribonucleosides and is not restricted tothe introduction of protecting groups as described above. The methodsfor the preparation of protected ribonucleotides currently known in theart, such as the organometallic route, are not 2′-3′ selective for theintroduction of the protecting group. This method allows the selectiveintroduction of the methylthiomethyl group at the 2′ position andthereby prevents an unselective step late in the synthesis scheme.

In this new route a 2′-O-alkythiomethyl-ribonucleoside as can berepresented by the formula

wherein R₇ is alkyl or aryl or a combination of alkyl and aryl, isreacted with a silanol of the general formula HOSiR₄R₅R₆. In a preferredembodiment R₇ is a (C₁ to C₂₀)-, more preferred is (C₁ to C₁₀)-alkyland/or aryl. In another preferred embodiment R₇ is for instance methyl,ethyl, propyl, butyl, pentyl, hexyl, iso-propyl, sec-butyl, iso-butyl,sec-pentyl. The substituents R₄, R₅ and R₆ of the silanol are identicalor different alkyl or aryl or a combination of alkyl and arylsubstituents, or substituted heteroatoms and which may also cyclicallybe connected to each other. In a preferred embodiment the threesubstituents together comprise between 3 and 30 carbon atoms each.

In another preferred embodiment, compound 11 is reacted with a silanolof the general formula HOSiR₄R₅R₆ with the substituents R₄, R₅, R₆ asdefined above, under suitable conditions known in the art. Thesecomprise an electrophilic activating reagent or reagent combination suchas, but not limited to, for instance N-halosuccinimides and a catalyticamount of an acid. The reaction may be carried out in solution or onsolid phase or by using polymer supported reagents. The solvent can be ahydrocarbon solvent, ethereal solvent, nitrile solvent, chlorinatedsolvent, heterocyclic solvent, sulfoxide solvents, etc. Specificexamples of suitable solvents include pyridine, N,N-dimethylformamide(DMF), tetrahydrofuran (THF), dimethylsulfoxide (DMSO), acetonitrile,dichloroethane and methylene chloride. Preferrably, dichloromethane isused. Although the reaction may be carried out at room temperature, itmay also be carried out at a temperature of −78° C. to 100° C.preferably at 0 to 50° C.

The alkylthiomethyl-substituent at the 2′-OH group comprises preferablya (C₁ to C₁₂)-alkyl and/or aryl group, preferably a (C₁ to C₆)-alkyland/or aryl group. Examples of preferred substituents comprisemethylthiomethyl, ethylthiomethyl, propylthiomethyl, isopropylthiomethylor butylthiomethyl.

The following Examples illustrate the present invention, without in anyway limiting the scope thereof.

EXAMPLES

1. Preparation of THEX Building Blocks via Organometallic Route

Scheme 1 depicts the synthetic scheme for the introduction of the THEXprotecting group on 5′-O-DMTr Uridine and the subsequentphosphitylation.

Preparation of (1,1,2-trimethyl-propyl)-dimethylsilyloxymethyl chloride(THEX—Cl)

A suspension of 11.1 ml (0.15 mol) ethanethiol and 4.5 g (0.15 mol)para-formaldehyde was treated with two drops of NaOMe/MeOH (30%) andstirred 1 h at 40° C. After cooling, 150 ml CH₂Cl₂ and 22.66 g (0.333mol) imidazole were added. After 10 minutes, 32.66 g (0.1 67 mol)(1,1,2-trimethyl-propyl)-dimethylsilyl chloride was added dropwise. Theresulting suspension was stirred at room temperature for 24 hours anddiluted with 300 ml n-hexane. After adding 200 ml 2M NaH₂PO₄ solution,stirring (15 minutes) and phase separation, the organic phase was driedover Na₂SO₄ and evaporated. The residue was dissolved in 100 ml CH₂Cl₂,treated dropwise with 12.3 ml (20.4 g, 0.152 mol) sulfurylchloride in 50ml CH₂Cl₂. After 1 hour, the mixture was evaporated. The product wasobtained (31.5 g) as wax.

¹H-NMR (400 MHz, CDCl₃): 0.5 (s,6H,SiMe₂); 0.65 (12H,CH₃); 1.40(sept,1H,CH); 5.43 (s,2H,CH₂).

Preparation of1-[5′-O-(4,4′-Dimethoxytrityl)-2′-O-[((1,1,2-trimethyl-propyl)-dimethylsilyl)]-oxymethyl-beta-D-ribofuranosyl]-uracil(5a)

A solution of 9.5 g (17.4 mmol) 5′-O-dimethoxytritylated uridine(1) in200 ml 1,2-dichloroethane was treated with 11.23 g (87 mmol) Huenig'sbase and then with 5.81 g (19.2 mmol) dibutyl-tindichloride. After 30minutes, the mixture was heated to 80° C., treated with 4.2 g (22.6mmol) (1,1,2-trimethyl-propyl)-dimethylsilyloxymethyl chloride (THEX—Cl)in 50 ml dichloroethane and stirred two hours at 80° C. After cooling,the mixture was diluted with 400 ml CH₂Cl₂ and 350 ml aqueous saturatedNaHCO₃ solution were added. After stirring 30 minutes, the layers wereseparated and the organic layer was evaporated. The residue waschromatographed on silica gel, using ethylacetate/hexane(3:1) containing0.1% N-methylmorpholine. The product was obtained as solid foam (4.52g).

¹H-NMR (400 MHz, CDCl₃): 0.1 (s,6H,CH₃); 0.6-0.8 (s and d,12H,CH₃); 1.45(m,1H,CH); 3.15 (d,2H,CH₂); 3.64 (s,6H,OCH₃); 3.85 (q,2H,CH₂); 4.05(m,1H,CH); 4.15 (m,1H,CH); 4.80 (q,2H,CH₂); 5.12 (d,1H,OH); 5.30(q,1H,CH); 5.78 d,1H CH); 6.8-5.3 (m,13H); 7.6 (d.1H).

Preparation of1-[5′-O-(4,4′-Dimethoxytrityl)-2′-O-[((1,1,2-trimethyl-propyl)-dimethylsilyl)]-oxymethyl-beta-D-ribofuranosyl]-uracil-3′-(2-cyanoethyldiisopropyl)phosphoramidite(6)

A solution of 4.0 g (5.56 mmol) protected uridine(2), 1.14 g (6.68 mmol)diisopropylaminotetrazolid and 2.01 g (6.68 mmol)bis(N,N-diisopropylamino)-2-cyanoethoxy phosphine in 150 ml CH₂Cl₂ werestirred 24 hours at room temperatur. The mixture was diluted with 100 mlCH₂Cl₂ and washed twice with 50 ml aqueous saturated NaHCO₃ solution.The dried (Na₂SO₄) organic phase was evaporated and the residue wassubjected to column chromatography (ethylacetate/hexane 3:2 withadditional 0.1% N-methylmorpholine). The product was obtained as a solidfoam (4.12 g).

³¹P-NMR (400 MHz, CDCl₃): 151.183 (s) and 151.537 (s).

2. Preparation of THEX Building Blocks via 2′-O-methylthiomethyl Route

3′,5′-O-Diacetyl-2′-O-methylthiomethyl-uridine(8)

A solution of 4.53 g (14.8 mmol) 2′-O-methylthiomethyl uridine 7 (8) in50 ml pyridin was treated with 3.04 g (29.7 mmol) Ac₂O. After 24 hstirring, the solution was evaporated. The residue was dissolved in 40ml EtOAc, washed with water and dried with Na₂SO₄. After evaporation,the pure title-compound was obtained (5.05 g).

3′,5′-O-Diacetyl-2′-O-[((1,1,2-trimethyl-propyl)-dimethylsilyl)-oxymethyl]-uridine(9)

0.33 g (1.47 mmol) N-iodosuccinimid in 3 ml THF was added to a solutionof 0.5 g (1.287 mmol) 8, 0.978 g (6.1 mmol)(1,1,2-trimethyl-propyl)-dimethyl silanol, 10 ml CH₂Cl₂ and 1 dropMeOSO₃H. After 2 h stirring, 2 ml NaHSO₃ (37%) was added, then 100 mlCH₂Cl₂. The organic layer was separated and dried with Na₂SO₄. Afterfiltration, the solution was evaporated: 552 mg pure title-compound.

2′-O-[((1,1,2-trimethyl-propyl)-dimethylsilyl)-oxymethyl]-uridine(10)

A solution of 187 mg (0.37 mmol) 9, 20 ml MeOH and 0.135 ml (0.74 mmol)NaOMe/MeOH (30%) was stirred 30 min. at 0° C. After evaporation, theresidue was filtered through a small silica gel column (EtOAc/MeOH 4:1):140 mg 10 as powder.

3. Procedure for the Incorporation of 6 into Oligodeoxynucleotide byPhosphoramidite Chemistry and Fast Deprotection Thereof

Oligonucleotide synthesis were typically performed on an ABI394automated DNA synthesizer (Applied Biosystems). DNA Phosphoramidites,THEX protected Uridine phosphoramidite (6) or TOM protected Uridinephosphoramidite (Xeragon, Inc.) were dissolved in dry acetonitrile at a5% w/v concentration; coupling was made by activation ofphosphoramidites using a 0.2 M solution of benzimidazolium triflate (9)in acetonitrile. Coupling times were between 1-5 minutes. A firstcapping was made using standard capping reagents. Oxidation was madeusing an 0.1M iodine solution in THF/water/pyridine (1:1:1). A secondcapping was performed after oxidation. Detritylation before the nextcoupling was effected with 2% dichloroacetic acid in dichloroethane.

Upon completion of oligonucleotide chain elongation, the solid supportwas transferred to an Eppendorf tube.

When prepared with THEX protected Uridine phosphoramidite (6),oligonucleotides were cleaved from support and deprotected as follows:

-   -   1. 32% aq. Ammonia/EtOH 3:1 (250 μl for 0.2 μmole scale), room        temperature, 2 h lyophilisation to dryness.    -   2. 1M tetrabutylammonium fluoride in THF (250 μl for 0.2 μmole        scale), 30 min at room temperature.    -   3. 1M Tris.HCl, pH=7.4 (250 μl for 0.2 μmole scale).

When prepared with TOM protected Uridine phosphoramidite,oligonucleotides were cleaved from support and deprotected as follows:

-   -   1. 32% aq. Ammonia/EtOH 3:1 (250 μl for 0.2 μmole scale), room        temperature, 2 h lyophilisation to dryness.    -   2. 1M tetrabutylammonium fluoride in THF (250 μl for 0.2 μmole        scale), 6 h min at room temperature.    -   3. 1M Tris.HCl, pH=7.4 (250 μl for 0.2 μmole scale).

Resulting crude solutions were analysed by Capillary GelElectrophoresis.

Results are summarized in table 1 TOM THEX # Sequence (purity %) (purity%) 11 TTT TTU TTT TTT TTT 85 79 12 TTT TTU UUU TTT TTT 67 72

As shown in table 1, and FIGS. 1-4, quality of crude material obtainedwith THEX protected Uridine phosphoramidite 6 and TOM protected Uridinephosphoramidite are very similar. Use of 2′-O-THEX protecting groupstrategy allowed the reduction of 2′ deprotection from 6 h at 35° C. (asreported in ref. 6) to 30 min at room temperature.

REFERENCES

-   1. De Mesmaeker, A., Haener, R., Martin, P., Moser, H. E. Acc. Chem.    Res. 28 (1995) 366 and Bennett, C. Frank, Cowsert, Lex M. Curr.    Opin. Mol. Ther. 1, (1999), 359.-   2. Elbashir, Sayda M.; Harborth, Jens; Lendeckel, Winfried; Yalcin,    Abdullah; Weber, Klaus; Tuschl, Thomas. Duplexes of 21-nucleotide    RNAs mediate RNA interference in cultured mammalian cells. Nature    (London, United Kingdom) (2001), 411(6836), 494-498.-   3. Usman, N.; Ogilvie, K. K.; Jiang, M. Y.; Cedergren, R. J. The    automated chemical synthesis of long oligoribuncleotides using    2′-O-silylated ribonucleoside 3′-O-phosphoramidites on a    controlled-pore glass support: synthesis of a 43-nucleotide sequence    similar to the 3′-half molecule of an Escherichia coli    formylmethionine tRNA. J. Am. Chem. Soc. (1987), 109(25), 7845-54.-   4. Morgan, Michael A.; Kazakov, Sergei A.; Hecht, Sidney M.    Phosphoryl migration during the chemical synthesis of RNA. Nucleic    Acids Research (1995), 23(19), 3949-53.-   5. Welz, Rudiger; Muller, Sabine. 5-(Benzylmercapto)-1H-tetrazole as    activator for 2′-O-TBDMS phosphoramidite building blocks in RNA    synthesis. Tetrahedron Letters (2002), 43(5), 795-797-   6. Pitsch, Stefan; Weiss, Patrick A.; Jenny, Luzi; Stutz, Alfred;    Wu, Xiaolin. Reliable chemical synthesis of oligoribonucleotides    (RNA) with 2′-O-[(triisopropylsilyl)oxy]methyl(2′-O-tom)-protected    phosphoramidites. Helvetica Chimica Acta (2001), 84(12), 3773-3795.-   7. Gundersen, Lise Lotte; Benneche, Tore; Undheim, Kjell.    Chloromethoxysilanes as protecting reagents for sterically hindered    alcohols. Acta Chem. Scand. (1989), 43(7), 706-9.-   8. Russian J. Bioorg. Chem. 2000,26,327-333.-   9. Hayakawa, Yoshihiro; Kataoka, Masanori; Noyori, Ryoji.    Benzimidazolium Triflate as an Efficient Promoter for Nucleotide    Synthesis via the Phosphoramidite Method. Journal of Organic    Chemistry (1996), 61(23), 7996-7997.

1. A ribonucleoside-derivative of the formula

wherein R₁ is a base of the purine- or pyrimidine-family or a derivativeof such a base or any other residue which serves as a nucleobasesurrogate, R₂ is a proton or a substituted derivative of phosphoricacid, R₃ is a proton or a protection-group for the oxygen atom in5′-position, R₄, R₅ and R₆ are independently alkyl or aryl or acombination of alkyl and aryl or heteroatom, R₄, R₅ or R₆ may also becyclically connected to each other; and wherein at least one of the R₄,R₅ or R₆ substituents comprises a tertiary C-atom or a heteroatomvicinal to the Si-atom.
 2. A ribonucleoside-derivative according toclaim 1 wherein the substituent comprising the tertiary C-atom vicinalto the Si-atom comprises from 4 to 24 C-atoms.
 3. Aribonucleoside-derivative according to claim 1 wherein the substituentcomprising the tertiary C-atom vicinal to the Si-atom is analkyl-substituent selected from the group consisting of tert-butyl,tert-pentyl, tert-hexyl, tert-heptyl, tert-octyl, tert-nonyl,tert-decyl, tert-undecyl, tert-dodecyl.
 4. A ribonucleoside-derivativeaccording to claim 1 wherein the substituent comprising the tertiaryC-atom vicinal to the Si-atom is selected from the group of 1,1-dimethylethyl, 1,1-dimethyl-propyl, 1,1-dimethyl-butyl, 1,1-dimethyl-pentyl,1,1-dimethyl-hexyl, 1,1,2-trimethyl-propyl, 1,1,2-trimethyl-butyl,1,1,2-trimethyl-pentyl, 1,1,2-trimethyl-hexyl,1,1,2,2tetramethyl-propyl, 1,1,2,2-tetramethyl-butyl.
 5. Aribonucleoside-derivative according to claim 1 wherein the substituentvicinal to the Si-atom comprises a substituted heteroatom.
 6. Aribonucleoside-derivative according to claim 5 wherein the substituentvicinal to the Si-atom comprises a substituted bivalent heteroatom.
 7. Aribonucleoside-derivative according to claim 6 wherein the heteroatom isoxygen.
 8. A method for the preparation of a ribonucleoside-derivativeaccording to claim 1, comprising reacting a nucleoside with the formula

where R₁ and R₃ are as defined in claim 1, with asilyloxymethyiderivative of the formula

wherein Y is a suitable leaving group and wherein R₄, R₅ and R₆ areindependently alkyl or aryl or a combination of alkyl and aryl or aheteroatom, R₄, R₅ or R₆ may also be cyclically connected to each other.9. The method of claim 8 wherein Y is a halogen.
 10. The method of claim8 wherein R₄, R₅ and R₆ together comprise between 3 and 30 carbon atoms.11. The method of claims 8 wherein R₄, R₅ or R₆ comprise at least onesubstituted heteroatom vicinal to Si atom.
 12. The method of claim 11wherein the heteroatom is a bivalent atom.
 13. The method of claim 12wherein the heteroatom is oxygen.
 14. The method of claim 11 wherein theribonucleoside derivative is further substituted on the oxygen in3′-position with a group comprising of a derivative of phosphoric acid.15. A method for the preparation of a ribonucleoside-derivative,comprising reacting a ribonucleoside derivative with the formula

upon an electrophilic activation with a compound of formula:

wherein R₁ is defined as in claim 1 and R₇ is a alkyl- or aryl-group, oralkyl-aryl-group, wherein R₂ is a protecting group, wherein R₃ is aprotecting group, wherein R₄, R₅ and R₆ are identical or different alkylor aryl or a combination of alkyl and aryl substituents, which my befurther substituted with heteroatoms and which may also cyclically beconnected to each other.
 16. The method of claim 15 wherein R₄, R₅ andR₆ are defined as in claims
 1. 17. The method of claim 15 wherein theribonucleoside derivative is further substituted on the oxygen in3′-position with a group comprising of a derivative of phosphoric acid.